The Hha-TomB toxin-antitoxin module in Salmonella enterica serovar Typhimurium limits its intracellular survival profile and regulates host immune response.

The key to bacterial virulence relies on an exquisite balance of signals between microbe and hosts. Bacterial toxin-antitoxin (TA) system is known to play a vital role in response to stress adaptation, drug resistance, biofilm formation, intracellular survival, persistence as well as pathogenesis. In the present study, we investigated the role of Hha-TomB TA system in regulating virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium) in a host model system, where we showed that deletion of hha and tomB genes displayed impaired cell adhesion, invasion, and uptake. The isogenic hha and tomB mutant strain was also found to be deficient in intracellular replication in vitro, with a highly repressed Salmonella Pathogenicity Island-2 (SPI-2) genes and downregulation of Salmonella Pathogenicity Island-1 (SPI-1) genes. In addition, the Δhha and ΔtomB did not show acute colitis in C57BL/6 mice and displayed less dissemination to systemic organs followed by their cecal pathology. The TA mutants also showed reduction in serum cytokine and nitric oxide levels both in vitro and in vivo. However, the inflammation phenotype was restored on complementing strain of TA gene to its mutant strain. In silico studies depicted firm interaction of Hha-TomB complex and the regulatory proteins, namely, SsrA, SsrB, PhoP, and PhoQ. Overall, we demonstrate that this study of Hha-TomB TA system is one of the prime regulating networks essential for S. Typhimurium pathogenesis. 1. Role of Hha-TomB toxin-antitoxin (TA) system in Salmonella pathogenesis was examined. 2. The TA mutants resulted in impaired invasion and intracellular replication in vitro. 3. The TA mutants displayed alteration in SPI-1 and SPI-2 regulatory genes inside host cells. 4. Mutation in TA genes also limited systemic colonization and inflammatory response in vivo.

Marine Antimicrobial Peptides-Based Strategies for Tackling Bacterial Biofilm and Biofouling Challenges.

An assemblage nexus of microorganisms enclosed in a composite extracellular polymeric matrix is called as a biofilm. The main factor causing biological fouling, or biofouling, is biofilms. Biofilm-mediated biofouling is a significant detrimental issue in several industries, including the maritime environment, industrial facilities, water treatment facilities, and medical implants. Conventional antibacterial remedies cannot wholly eradicate bacterial species owing to the structural rigidity of biofilm and the eventual growth of antibiotic-resistant microorganisms. Consequently, several approaches to disrupt the biofilm have been investigated to address this particular phenomenon. Antimicrobial peptides (AMPs) have emerged as a promising contender in this category, offering several advantages over traditional solutions, including broad-spectrum action and lack of antibiotic resistance. Because biofouling significantly impacts the marine industry, AMPs derived from marine sources may be suitable natural inhibitors of bacterial proliferation. In this article, we discuss the range of physicochemical and structural diversity and the model of action seen in marine AMPs. This makes them an appealing strategy to mitigate biofilm and biofilm-mediated biofouling. This review also systematically summarizes recent research on marine AMPs from vertebrates and invertebrates and their industrial significance, shedding light on developing even better anti-biofouling materials shortly.

B and T cell epitope-based peptides predicted from clumping factor protein of Staphylococcus aureus as vaccine targets.

Staphylococcus aureus infection is emerging as a global threat because of the highly debilitating nature of the associated disease's unprecedented magnitude of its spread and growing global resistance to antimicrobial medicines. Recently WHO has categorized these bacteria under the high global priority pathogen list and is one of the six nosocomial pathogens termed as ESKAPE pathogens which have emerged as a serious threat to public health worldwide. The development of a specific vaccine can stimulate an optimal antibody response, thus providing immunity against it. Therefore, in the present study efforts have been made to identify potential vaccine candidates from the Clumping factor surface proteins (ClfA and ClfB) of S. aureus. Employing the immunoinformatics approach, fourteen antigenic peptides including T-cell, B-cell epitopes were identified which were non-toxic, non-allergenic, high antigenicity, strong binding efficiency with commonly occurring MHC alleles. Consequently, a multi-epitope vaccine chimera was designed by connecting these epitopes with suitable linkers an adjuvant to enhance immunogenicity. Further, homology modeling and molecular docking were performed to construct the three-dimensional structure of the vaccine and study the interaction between the modeled structure and immune receptor (TLR-2) present on lymphocyte cells. Consequently, molecular dynamics simulation for 100 ns period confirmed the stability of the interaction and reliability of the structure for further analysis. Finally, codon optimization and in silico cloning were employed to ensure the successful expression of the vaccine candidate. As the targeted protein is highly antigenic and conserved, hence the designed novel vaccine construct holds potential against emerging multi-drug-resistant organisms.

Genome-based identification and comparative analysis of enzymes for carotenoid biosynthesis in microalgae.

Microalgae are potential feedstocks for the commercial production of carotenoids, however, the metabolic pathways for carotenoid biosynthesis across algal lineage are largely unexplored. This work is the first to provide a comprehensive survey of genes and enzymes associated with the less studied methylerythritol 4-phosphate/1-deoxy-D-xylulose 5-phosphate pathway as well as the carotenoid biosynthetic pathway in microalgae through bioinformatics and comparative genomics approach. Candidate genes/enzymes were subsequently analyzed across 22 microalgae species of lineages Chlorophyta, Rhodophyta, Heterokonta, Haptophyta, Cryptophyta, and known Arabidopsis homologs in order to study the evolutional divergence in terms of sequence-structure properties. A total of 403 enzymes playing a vital role in carotene, lutein, zeaxanthin, violaxanthin, canthaxanthin, and astaxanthin were unraveled. Of these, 85 were hypothetical proteins whose biological roles are not yet experimentally characterized. Putative functions to these hypothetical proteins were successfully assigned through a comprehensive investigation of the protein family, motifs, intrinsic physicochemical features, subcellular localization, pathway analysis, etc. Furthermore, these enzymes were categorized into major classes as per the conserved domain and gene ontology. Functional signature sequences were also identified which were observed conserved across microalgal genomes. Additionally, the structural modeling and active site architecture of three vital enzymes, DXR, PSY, and ZDS catalyzing the vital rate-limiting steps in Dunaliella salina were achieved. The enzymes were confirmed to be stereochemically reliable and stable as revealed during molecular dynamics simulation of 100 ns. The detailed functional information about individual vital enzymes will certainly help to design genetically modified algal strains with enhanced carotenoid contents.

Structure of an Unfolding Intermediate of an RRM Domain of ETR-3 Reveals Its Native-like Fold.

Prevalence of one or more partially folded intermediates during protein unfolding with different secondary and ternary conformations has been identified as an integral character of protein unfolding. These transition-state species need to be characterized structurally for elucidation of their folding pathways. We have determined the three-dimensional structure of an intermediate state with increased conformational space sampling under urea-denaturing condition. The protein unfolds completely at 10 M urea but retains residual secondary structural propensities with restricted motion. Here, we describe the native state, observable intermediate state, and unfolded state for ETR-3 RRM-3, which has canonical RRM fold. These observations can shed more light on unfolding events for RRM-containing proteins.

Interaction studies on bacterial stringent response protein RelA with uncharged tRNA provide evidence for its prerequisite complex for ribosome binding.

The bacterial stringent response is regulated by the synthesis of (p)ppGpp which is mediated by RelA in a complex with uncharged tRNA and ribosome. We intended to probe RelA-uncharged tRNA interactions off the ribosome to understand the sequential activation mechanism of RelA. Stringent response is a key regulatory pleiotropic mechanism which allows bacteria to survive in unfavorable conditions. Since the discovery of RelA, it has been believed that it is activated upon binding to ribosomes which already have uncharged tRNA on acceptor site (A-site). However, uncharged tRNA occupied in the A-site of the ribosome prior to RelA binding could not be observed; therefore, recently an alternate model for RelA activation has been proposed in which RelA first binds to uncharged tRNA and then RelA-uncharged tRNA complex is loaded on to the ribosome to synthesize (p)ppGpp. To explore the alternate hypothesis, we report here the in vitro binding of uncharged tRNA to RelA in the absence of ribosome using formaldehyde cross-linking, fluorescence spectroscopy, surface plasmon resonance, size-exclusion chromatography, and hydrogen-deuterium exchange mass spectrometry. Altogether, our results clearly indicate binding between RelA and uncharged tRNA without the involvement of ribosome. Moreover, we have analyzed their binding kinetics and mapping of tRNA-interacting regions of RelA structure. We have also co-purified TGS domain in complex with tRNA to further establish in vivo RelA-tRNA binding. We have observed that TGS domain recognizes all types of uncharged tRNA similar to EF-Tu and tRNA interactions. Altogether, our results demonstrate the complex formation between RelA and uncharged tRNA that may be loaded to the ribosome for (p)ppGpp synthesis.

Structural and functional insights into peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase is an essential enzyme which acts as one of the rescue factors of the stalled ribosomes. It is an esterase that hydrolyzes the ester bond in the peptidyl-tRNA molecules, which are products of ribosome stalling. This enzyme is required for rapid clearing of the peptidyl-tRNAs, the accumulation of which in the cell leads to cell death. Over the recent years, it has been heralded as an attractive drug target for antimicrobial therapeutics. Two distinct classes of peptidyl-tRNA hydrolase, Pth and Pth2, have been identified in nature. This review gives an overview of the structural and functional aspects of Pth, along with its sequence and structural comparison among various species of bacteria. While the mode of binding of the substrate to Pth and the mechanism of hydrolysis are still speculated upon, the structure-based drug design using this protein as the target is still largely unexplored. This review focuses on the structural features of Pth, giving a direction to structure-based drug design on this protein.

Current overview of allergens of plant pathogenesis related protein families.

Pathogenesis related (PR) proteins are one of the major sources of plant derived allergens. These proteins are induced by the plants as a defense response system in stress conditions like microbial and insect infections, wounding, exposure to harsh chemicals, and atmospheric conditions. However, some plant tissues that are more exposed to environmental conditions like UV irradiation and insect or fungal attacks express these proteins constitutively. These proteins are mostly resistant to proteases and most of them show considerable stability at low pH. Many of these plant pathogenesis related proteins are found to act as food allergens, latex allergens, and pollen allergens. Proteins having similar amino acid sequences among the members of PR proteins may be responsible for cross-reactivity among allergens from diverse plants. This review analyzes the different pathogenesis related protein families that have been reported as allergens. Proteins of these families have been characterized in regard to their biological functions, amino acid sequence, and cross-reactivity. The three-dimensional structures of some of these allergens have also been evaluated to elucidate the antigenic determinants of these molecules and to explain the cross-reactivity among the various allergens.

Structural and Biochemical Studies on Klebsiella Pneumoniae Enoyl-ACP Reductase (FabI) Suggest Flexible Substrate Binding Site.

Klebsiella pneumoniae, a bacterial pathogen infamous for antibiotic resistance, is included in the priority list of pathogens by various public health organizations due to its extraordinary ability to develop multidrug resistance. Bacterial fatty acid biosynthesis pathway-II (FAS-II) has been considered a therapeutic drug target for antibacterial drug discovery. Inhibition of FAS-II enzyme, enoyl-acyl carrier protein reductase, FabI, not only inhibits bacterial infections but also reverses antibiotic resistance. Here, we characterized Klebsiella pneumoniae FabI (KpFabI) using complementary experimental approaches including, biochemical, x-ray crystallography, and molecular dynamics simulation studies. Biophysical studies shows that KpFabI organizes as a tetramer molecular assembly in solution as well as in the crystal structure. Enzyme kinetics studies reveal a distinct catalytic property towards crotonyl CoA and reducing cofactor NADH. Michaelis-Menten constant (Km) values of substrates show that KpFabI has higher preference towards NADH as compared to crotonyl CoA. The crystal structure of tetrameric apo KpFabI folds into a classic Rossman fold in which ß-strands are sandwiched between α-helices. A highly flexible substrate binding region is located toward the interior of the tetrameric assembly. Thermal stability assay on KpFabI with its substrate shows that the flexibility is primarily stabilized by cofactor NADH. Moreover, the molecular dynamics further supports that KpFabI has highly flexible regions at the substrate binding site. Together, these findings provide evidence for highly dynamic substrate binding sites in KpFabI, therefore, this information will be vital for specific inhibitors discovery targeting Klebsiella pneumoniae.

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states.

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67Å, 1.60Å, 2.20Å, 1.70Å, 2.07Å and 1.90Å resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.

First structural evidence of sequestration of mRNA cap structures by type 1 ribosome inactivating protein from Momordica balsamina.

This is the first structural evidence of recognition of mRNA cap structures by a ribosome inactivating protein. It is well known that a unique cap structure is formed at the 5' end of mRNA for carrying out various processes including mRNA maturation, translation initiation, and RNA turnover. The binding studies and crystal structure determinations of type 1 ribosome inactivating protein (RIP-1) from Momordica balsamina (MbRIP-1) were carried out with mRNA cap structures including (i) N7-methyl guanine (m7G), (ii) N7-methyl guanosine diphosphate (m7GDP), and (iii) N7-methyl guanosine triphosphate (m7GTP). These compounds showed affinities to MbRIP-1 at nanomolar concentrations. The structure determinations of the complexes of MbRIP-1 with m7G, m7GDP, and m7GTP at 2.65, 1.77, and 1.75 Å resolutions revealed that all the three compounds bound to MbRIP-1 in the substrate binding site at the positions which are slightly shifted towards Glu85 as compared to those of rRNA substrates. In this position, Glu85 forms several hydrogen bonds with guanine moiety while N-7 methyl group forms van der Waals contacts. However, the guanine rings are poorly stacked in these complexes. Thus, the mode of binding by MbRIP-1 to mRNA cap structures is different which results in the inhibition of depurination. Since some viruses are known to exploit the capping property of the host, this action of MbRIP-1 may have implications for the antiviral activity of this protein in vivo. The understanding of the mode of binding of MbRIP-1 to cap structures may also assist in the design of anti-viral agents.

Genome analysis and virulence gene expression profile of a multi drug resistant Salmonella enterica serovar Typhimurium ms202.

BACKGROUND: In India, multi-drug resistance in Salmonella enterica serovar Typhimurium poses a significant health threat. Indeed, S. Typhimurium has remained unknown for a large portion of its genome associated with various physiological functions including mechanism of drug resistance and virulence. The whole-genome sequence of a Salmonella strain obtained from feces of a patient with gastroenteritis in Odisha, India, was analyzed for understanding the disease association and underlying virulence mechanisms. RESULTS: The de novo assembly yielded 17 contigs and showed 99.9% similarity to S. enterica sub sp enterica strain LT2 and S. enteric subsp salamae strain DSM 9220. S. Typhimurium ms202 strain constitutes six known Salmonella pathogenicity islands and nine different phages. The comparative interpretation of pathogenic islands displayed the genes contained in SPI-1 and SPI-2 to be highly conserved. We identified sit ABCD cluster regulatory cascade in SPI-1. Multiple antimicrobial resistance genes were identified that directly implies antibiotic-resistant phenotype. Notably, seven unique genes were identified as "acquired antibiotic resistance". These data suggest that virulence in S. enterica Typhimurium ms202 is associated with SPI-1 and SPI-2. Further, we found several virulent genes encoding SPI regions belonging to type III secretion systems (T3SS) of bacteria were significantly upregulated in ms202 compared to control LT2. Moreover, all these genes were significantly downregulated in S. enterica Typhimurium ms202 as compared to control LT2 on adding Mn2+ exogenously. CONCLUSIONS: Our study raises a vital concern about the potential diffusion of a novel multi-drug resistant S. enterica Typhimurium ms202. It justifies this clinical pathogen to demonstrate a higher degree survival due to higher expression of virulent genes and enhanced ability of metallic ion acquisition.

Structural investigation on SPI-6-associated Salmonella typhimurium VirG-like stress protein that promotes pathogen survival in macrophages.

Enteric microbial pathogenesis, remarkably a complex process, is achieved by virulence factors encoded by genes located within regions of the bacterial genome termed pathogenicity islands. Salmonella pathogenicity islands (SPI) encodes proteins, that are essential virulence determinants for pathogen colonization and virulence. In addition to the well-characterized SPI-1 and SPI-2 proteins, which are required for bacterial invasion and intracellular replication, respectively, SPI-6 (formerly known as Salmonella enterica centisome 7 island [SCI]) encoding proteins are also known to play pivotal role in Salmonella pathogenesis. However, the underlying molecular mechanism of these proteins remained elusive. To gain molecular insights into SPI-6-associated proteins, in this study, a SPI-6 Salmonella typhimurium VirG-like protein (STV) is characterized using interdisciplinary experimental approaches including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and infection assays. The high-resolution crystal structure, determined by the single-wavelength anomalous dispersion (SAD) method, reveals that STV belongs to the LTxxQ motif family. Solution-state NMR spectroscopy studies reveal that STV form a dimer involving interconnected helices. Interestingly, functional studies show that STV influence pathogen persistence inside macrophages in vitro at later stages of infection. Altogether, our findings suggest that STV, a member of the LTxxQ stress protein family, modulates bacterial survival mechanism in macrophages through SPI-1 and SPI-2 genes, respectively.

Immunoinformatic approach employing modeling and simulation to design a novel vaccine construct targeting MDR efflux pumps to confer wide protection against typhoidal Salmonella serovars.

Overcoming multi drug resistance is one of the crucial challenges to control enteric typhoid fever caused by Salmonella typhi and Salmonella paratyphi. Overexpression of efflux pumps predominantly causes drug resistance in microorganisms. Therefore, immunotherapy targeting the various efflux pumps antigens could be a promising strategy to increase the success of vaccines. An immunoinformatic approach was employed to design a Salmonellosis multi-epitope subunit vaccine peptide consisting of linear B-cell and T-cell epitopes of multidrug resistance protein families including ATP Binding Cassette (ABC), major facilitator superfamily (MFS), resistance nodulation cell division (RND), small multidrug resistance (SMR), and multidrug and toxin extrusion (MATE). The selected epitopes exhibited conservation in both S. typhi and S. paratyphi and thus could be helpful for cross-protection. Further, the final vaccine construct encompassing the peptides, adjuvants and specific linker sequences showed high immunogenicity, solubility, non-allergenic, nontoxic, and wide population coverage due to strong binding affinity to maximum HLA alleles. The three-dimensional structure was predicted, and validated using various structure validation tools. Additionally, protein-protein docking of the chimeric vaccine construct with the TLR-2 protein and molecular dynamics demonstrated stable and efficient binding. Conclusively, the immunoinformatic study showed that the novel multi epitopic vaccine construct can simulate the both T-cell and B-cell immune responses in typhoidal Salmonella serovars and could potentially be used for prophylactic or therapeutic applications.Communicated by Ramaswamy H. Sarma.

Molecular Characterization and Designing of a Novel Multiepitope Vaccine Construct Against Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa, an ESKAPE pathogen causes many fatal clinical diseases in humans across the globe. Despite an increase in clinical instances of Pseudomonas infection, there is currently no effective vaccine or treatment available. The major membrane protein candidate of the P. aeruginosa bacterial cell is known to be a critical component for cellular bacterial susceptibility to antimicrobial peptides and survival inside the host organisms. Therefore, the current computational study aims to examine P. aeruginosa's major membrane protein, OprF, and OprI, in order to design linear B-cell, cytotoxic T-cell, and helper T-cell peptide-based vaccine constructs. Utilizing various immune-informatics tools and databases, a total of two B-cells and twelve T-cells peptides were predicted. The final vaccine design was simulated to generate a high-quality three-dimensional structure, which included epitopes, adjuvant, and linkers. The vaccine was shown to be nonallergenic, antigenic, soluble, and had the best biophysical properties. The vaccine and Toll-like receptor 4 have a strong and stable interaction, according to protein-protein docking and molecular dynamics simulations. Additionally, in silico cloning was employed to see how the developed vaccine expressed in the pET28a (+) vector. Ultimately, an immune simulation was performed to see the vaccine efficacy. In conclusion, the newly developed vaccine appears to be a promising option for a vaccine against P. aeruginosa infection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10989-021-10356-z.

Immunoinformatics and molecular docking studies reveal a novel Multi-Epitope peptide vaccine against pneumonia infection.

Pneumonia is a major endemic disease around the world, and an effective vaccine is the need of the hour to fight against the disease. When there are no appropriate antiviral and associated therapies available, vaccine development becomes even more essential. Therefore, in the present study, a variety of immunoinformatics techniques was utilized to develop a novel multi-epitope vaccine that targets the highly immunodominant type 3 fimbrial protein of Klebsiella pneumoniae, the causal organism for pneumonia. The putative B and T cell epitopes were predicted from the protein and screened for antigenicity, toxicity, allergenicity, and cross-reactivity with human proteomes. Subsequently, the selected epitopes were joined with the help of linkers to form a robust vaccine construct. In addition, an adjuvant was applied to the N-terminal of the construct to improve the immunogenicity of the vaccine. The physicochemical properties, solubility, the secondary and tertiary structure of the final vaccine were also established. MD simulations for 100 ns were employed to assess the stability of the vaccine-TLR-2 docked complex. The final vaccine was optimized and cloned in pET28a (+) vector with His-tag to achieve maximum vaccine protein expression for ease of purification. Immune simulation results indicated the potency of this vaccine candidate as a probable therapeutic agent. In conclusion, the overall results of various immunoinformatics tools and methods employed revealed that the constructed multi-epitope vaccine exhibits a high potential for stimulating both B and T-cells immune responses against pneumonia infection. However, experimental immunological studies are required to corroborate the viability of the novel multi-epitope construct as a commercial vaccine.

Development of a Conserved Chimeric Vaccine for Induction of Strong Immune Response against Staphylococcus aureus Using Immunoinformatics Approaches.

Staphylococcus aureus is one of the most notorious Gram-positive bacteria with a very high mortality rate. The WHO has listed S. aureus as one of the ESKAPE pathogens requiring urgent research and development efforts to fight against it. Yet there is a major layback in the advancement of effective vaccines against this multidrug-resistant pathogen. SdrD and SdrE proteins are attractive immunogen candidates as they are conserved among all the strains and contribute specifically to bacterial adherence to the host cells. Furthermore, these proteins are predicted to be highly antigenic and essential for pathogen survival. Therefore, in this study, using the immunoinformatics approach, a novel vaccine candidate was constructed using highly immunogenic conserved T-cell and B-cell epitopes along with specific linkers, adjuvants, and consequently modeled for docking with human Toll-like receptor 2. Additionally, physicochemical properties, secondary structure, disulphide engineering, and population coverage analysis were also analyzed for the vaccine. The constructed vaccine showed good results of worldwide population coverage and a promising immune response. For evaluation of the stability of the vaccine-TLR-2 docked complex, a molecular dynamics simulation was performed. The constructed vaccine was subjected to in silico immune simulations by C-ImmSim and Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells, and INF-γ. Lastly, upon cloning, the vaccine protein was reverse transcribed into a DNA sequence and cloned into a pET28a (+) vector to ensure translational potency and microbial expression. The overall results of the study showed that the designed novel chimeric vaccine can simultaneously elicit humoral and cell-mediated immune responses and is a reliable construct for subsequent in vivo and in vitro studies against the pathogen.

Structural Analysis of (p)ppGpp Reveals Its Versatile Binding Pattern for Diverse Types of Target Proteins.

(p)ppGpp, highly phosphorylated guanosine, are global regulatory nucleotides that modulate several biochemical events in bacterial physiology ranging from core central dogma to various metabolic pathways. Conventionally, (p)ppGpp collectively refers to two nucleotides, ppGpp, and pppGpp in the literature. Initially, (p)ppGpp has been discovered as a transcription regulatory molecule as it binds to RNA polymerase and regulates transcriptional gene regulation. During the past decade, several other target proteins of (p)ppGpp have been discovered and as of now, more than 30 proteins have been reported to be regulated by the binding of these two signaling nucleotides. The regulation of diverse biochemical activities by (p)ppGpp requires fine-tuned molecular interactions with various classes of proteins so that it can moderate varied functions. Here we report a structural dynamics of (p)ppGpp in the unbound state using well-defined computational tools and its interactions with target proteins to understand the differential regulation by (p)ppGpp at the molecular level. We carried out replica exchange molecular dynamics simulation studies to enhance sampling of conformations during (p)ppGpp simulation. The detailed comparative analysis of torsion angle conformation of ribose sugar of unbound (p)ppGpp and bound states of (p)ppGpp was carried out. The structural dynamics shows that two linear phosphate chains provide plasticity to (p)ppGpp nucleotides for the binding to diverse proteins. Moreover, the intermolecular interactions between (p)ppGpp and target proteins were characterized through various physicochemical parameters including, hydrogen bonds, van der Waal's interactions, aromatic stacking, and side chains of interacting residues of proteins. Surprisingly, we observed that interactions of (p)ppGpp to target protein have a consensus binding pattern for a particular functional class of enzymes. For example, the binding of (p)ppGpp to RNA polymerase is significantly different from the binding of (p)ppGpp to the proteins involved in the ribosome biogenesis pathway. Whereas, (p)ppGpp binding to enzymes involved in nucleotide metabolism facilitates the functional regulation through oligomerization. Analysis of these datasets revealed that guanine base-specific contacts are key determinants to discriminate functional class of protein. Altogether, our studies provide significant information to understand the differential interaction pattern of (p)ppGpp to its target and this information may be useful to design antibacterial compounds based on (p)ppGpp analogs.

Crystal structure of the usher chaperone YadV reveals a monomer with the proline lock in closed conformation suggestive of an intermediate state.

Cell surface pili assembled by the chaperone-usher (CU) pathway play a crucial role in the adhesion of uropathogenic Escherichia coli. YadV is the chaperone component of the CU pathway of Yad pili. Here, we report the crystal structure of YadV from E. coli. In contrast to major usher chaperones, YadV is a monomer in solution as well as in the crystallographic symmetry, and the monomeric form is a preferred state for interacting with pilus subunits. Moreover, we observed a closed conformation for the proline lock, a crucial structural element for chaperone-pilus subunit interaction. MD simulation shows that the closed state of the proline lock is not energetically stable. Thus, the structure of monomeric YadV with its closed proline lock may serve as an intermediate state to provide suitable access to pilus subunits.

Stringent response protein as a potential target to intervene persistent bacterial infection.

More than half of the world's population is infected with persistent bacterial infections, consequently, persisters are gradually becoming a major public health concern. During the persistent phase, bacterial pathogens deploy many regulatory strategies to compensate unfavorable host environmental conditions. The stringent response is one of such gene regulatory mechanisms which is stimulated by nutrient starvation. It is regulated by the synthesis of highly phosphorylated signaling nucleotides, (p)ppGpp or alarmone. (p)ppGpp is synthesized by ppGpp synthetases, and these proteins are classified as RelA/SpoT homolog (RSH) proteins. Subsequently, (p)ppGpp modulate several molecular and biochemical processes ranging from transcription to metabolism. Imperativeness of (p)ppGpp synthetases has been investigated by numerous approaches including microbiology and animal studies, thereby establishing that Rel enzyme deleted strains of pathogenic bacteria were unable to transform in persister form. In this review, we summarize recent findings to corroborate the rationality to consider (p)ppGpp synthetase as a potential target in discovering a novel class of antimicrobial agents to combat persistent infections. Moreover, inhibition studies on Mycobacterium tuberculosis (p)ppGpp synthetase shows that these inhibitors prevent dormant state transition and biofilm formation. Also, we have highlighted the structural biology of (p)ppGpp synthetases, which may provide significant information that could be used in structure-based inhibitor design.